LTP is triggered by Ca^* entry through the NMDAR; subsequently Ca^* activates calmodulin (CaM), which then activates CaMKIl. Despite extensive studies demonstrating the pivotal role of CaMKIl in LTP and memory, the mechanisms of its activation in living cells is not known. The goal ofthe proposed work is to understand these mechanisms in quantitative detail. This requires methods to measure biochemical events in single spines near the limit of optical resolution and a sophisticated modeling framework for simulating these reactions. Because the experimental and computational methods were not previously available, this will be the first attempt to account for the measured activation of an enzyme in a living cell. Aim 1. Measurements will be made of critical quantitative properties of the system, including free CaMKIl, CaM, Ng and CaMKIl. This will be done used calibrated optical methods. Aim 2. To model CaM activation requires information about the spatial/temporal gradients of Ca^* in spines. 2-photon uncaging of glutamate will be used to activate NMDARs in a controlled way; the resulting Ca^* elevation in the bulk spine cytoplasm will be measured. Aim 3. Using fluorescence lifetime methodology (FLIM)), the time course of CaMKIl activation in single spines will be measured. Computer simulations will then be used to predict how the elevation of Ca^*, as determined in Aim 2, leads to CaMKIl activation. The parameters determined in Aim 1 are needed for this calculation. This predicted CaMKIl activation will be compared to the measured activation. Aim 4. Neurogranin (Ng) is an abundant postsynaptic protein that binds CaM and may be important in controlling the CaM that is available to activate CaMKIl. The effects of Ng knockout will be studied. RELEVANCE (See instructions): The proposed research is relevant to addiction, which involves persistent changes in synaptic strength. The role of CaMKIl in the persistence of synaptic strength has recently been demonstrated; notably biochemical attack of CaMKIl has reversed synaptic strength. There is therefore the possiblity that agents that attack CaMKIl can be used to reverse the synaptic changes that underlie addiction.